The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Multiplex assays, i.e. methods for simultaneous detection or quantification of a multiple of analytes in a sample are as such well known. Such assays are assays that simultaneously measure multiple analytes in a single run. Multiplex assays can be classified based on how many analytes can be measured per assay: the amount of analytes range from a few (at least two) up a very high number. The commercial multiplex assays are typically designed for simultaneous detection of up to about 50 analytes. These methods can be used for analyses of nucleic acids and proteins, such as antibodies. Also carbohydrates and other chemical compounds can be measured.
The multiplex methods can be carried out in many alternative ways.
As an example can be mentioned a microarray which is a 2D array on a solid support that simultaneously assays a large number of biological analytes. In a protein microassay such as an antibody microassay different antibodies have been affixed on a solid support at separate locations in a predetermined pattern. These antibodies are used as capture molecules capable of capturing analytes (proteins) present in a sample.
As an example of a commercially available assay can be mentioned Luminex® xMAP Technology, which is a bead-based assay performed directly in a microtiter plate. Each assay contains a mixture of different microspheres (bead mix), where each bead type is defined by an individual fluorescent color tone for analyte classification and carries a specific capture reagent such as specific proteins (antibodies) on its surface. During incubation of the bead mix with the patient sample complementary reaction partner (antigens) bind to the capture antibodies on the micro-spheres. In a second incubation step the bound antigens are detected with labelled antibodies bearing a specific fluorescent marker. The amount of bound analyte (antigen) correlates directly to the fluorescent intensity of the detecting antibody allowing the quantification of analytes. The classification of the beads and the quantification of the antigens are performed with the Luminex analysis system, which is based on the technology of flow cytometry using two different lasers.
The use of multiple sequential biomarkers for diagnosis and prognosis of diseases has also been suggested.
Chirag R Parikh et al., Crit Care Med 2008 Vol. 36, No. 4 (Suppl); p S159-S165, suggests the use of multiple sequential biomarkers for assessing the duration of AKI (acute kidney injury) and for predicting overall prognosis with respect to dialysis requirement and mortality. The biomarkers were NGAL (neutrophil gelatinase-associated lipocalin) and cystatin C in a plasma panel and NGAL, IL-18 (interleukin-18) and KIM-1 (kidney injury molecule-1) in a urine panel.
O Beran et al., Eur J Clin Microbiol Infect Dis (2009) 28: 793-799 describes sequential analysis of biomarkers such as IL-6 (interleukin-6), IL-1ra (interleukin-1 receptor antagonist), IL-1beta (interleukin-1beta), IL-8 (interleukin-8), MIP-1beta (macrophage inflammatory protein-1beta) and MCP-1 (monocyte chemoattractant protein-1) and their correlation with IMD (invasive meningococcal disease) and the severity thereof.
WO 2009/053523, Faron Pharmaceuticals Oy, discloses that CD73 is a useful biomarker for monitoring the development of inflammatory diseases, in particular SIRS (systemic inflammatory response syndrome), ALI (acute lung injury), ARDS and MOF (multi-organ failure) in a patient. Tissue fluid samples were drawn from the patients at different points of time and the CD73 activity in the samples was determined. An increased level of CD73 activity was found to correlate with regression of the disease.
So far, nobody has suggested the use of multiple sequential biomarkers for monitoring the development of ARDS in a patient. Particularly, nobody has suggested the use of a set of biomarkers consisting of or including CD73 and IL-6 for this purpose.